We have continued our studies of the genetics and regulation of the biosynthesis of spermidine in E. coli, emphasizing the two enzymes involved, S-adenosylmethionine decarboxylase and spermidine synthase. We are particularly interested in S-adenosylmethionine decarboxylase, both because it is essential for spermidine synthesis and because it requires covalently-linked pyruvate for activity. We are also very interested in spermidine synthase, since no previous information was availabale on the genetics of this enzyme. In the current studies we have shown that the gene for spermidine synthase (speE) lies immediately adjacent to and upstream to the gene for S-adenosylmethionine decarboxylase (speD). Both genes are controlled by a single promoter upstream to the speE gene. Thus, these two genes form an operon and the genetic regulation of the speD gene is intimately dependent on the speE gene and its promoter. We have purified both proteins to homogeneity, and Dr. Darrell T. Liu (DBB, CDB) has carried out partial protein sequencing. We have also sequenced the DNA coding for these enzymes and now have shown, by the identity of the amino acid sequences deduced from the DNA sequences with those of the protein fragments, that each gene codes for the respective structural protein. We have shown that S-adenosylmethionine decarboxylase is formed as a proenzyme, which is then cleaved to produce two smaller fragments. We have identified the specific peptide bond that is cleaved to form the pyruvoyl group as a lysylserine linkage.